The major focus of this project is the measurement of the kinetic parameters of inhibition of bovine Factor VIIa, Factor IXa, Factor Xa, and thrombin by bovine antithrombin III in pure systems: specifically, the second-order rate constant for formation of the complex, its apparent dissociation constant, and the values of Km and kcat for any enzymatic cleavages of the complex that occur. Such a cleavage has been observed in the Factor Xa-antithrombin III complex. The activations of Factor X and prothrombin will be studied in pure systems in the presence of antithrombin III to show whether inhibitory control follows what will be predicted from the basic kinetic data. These activations will also be studied in plasma-based systems, to answer the questions of relative importance of control of coagulation by other plasma inhibitors. A brief study will be made of the mechanism of action of antithrombin III in the light of the known mechanism of the protease inhibitors from soybean and the pancreas: specifically, whether it is possible to generate an active two-chain inhibitor, and whether inhibition is competitive and reversible. Clinical observations clearly indicate the importance of antithrombin III in normal hemostasis. The proposed study aims to define quantitatively its action on the four enzymes involved in the middle and late stages of the clotting system. Such data are essential to an understanding of the kinetics of control of coagulation by inhibitors.